1. Field of the Invention
This invention relates generally to the method of determining the fluorescence threshold (noise level) of a flow cytometer relative qualitatively to the autofluorescence of cells and quantitatively in terms of the calibration units of molecules of equivalent soluble fluorochromes (MESF) using calibrated fluorescent microbead standards.
2. Description of the Related Art
Flow cytometers are used to analyze biological cells and particles in a fluid sample by intersecting a thin stream of fluid by an illumination source, usually a laser beam. The resulting forward and right angle scatter, and fluorescent light is analyzed with photomultiplier tubes (PMTs). The fluorescence channels of a flow cytometer, designated by FL1, FL2, FL3, etc. are each set with barrier filters to detect the selected specific dye while filtering out signals from other wavelengths.
In practice, however, these filters are not 100% effective in filtering out signals from other wavelengths and optical noise can arise from imperfections in the filters, pin-holes in the coatings, coating which themselves fluoresce, etc. Filters which are coated must be placed in the optical path such that the coating faces the correct direction, or else fluorescence from the coating will be passed along to the PMTs. In addition, small portions of the illumination source may be non-specifically scattered within the instrument and register in the PMTs due to the geometry of the instrument and optical path. These are some, but not all the optical sources of fluorescence noise which can reach the PMT as a signal.
Another source of noise resulting in a fluorescence signal comes the electronics of the flow cytometer, including, but not limited to, signal processors, the amplifiers, and the power source.
Measurements of all the parameters (forward scatter, side scatter, FL1, FL2, etc.) of individual cells or particles are taken when the instrument is triggered in a particular channel, usually the forward scatter. If a non-fluorescent particle triggers the forward scatter channel, theoretically, the instrument will be taking readings in the fluorescence channels (FL1, FL2, etc.) which are not from the particles themselves, but rather from the noise in each channel.
These sources of noise will be additive and the fluorescence threshold level of a particular fluorescence channel will be the summation of the optical and electronic noise, below which a fluorescence signal from a sample will not be possible to measure on that particular instrument.
Biological cells and many other particles have intrinsic fluorescence referred to as autofluorescence. Autofluorescence arises from fluorescent compounds or impurities within the cells or particles. For example, most mammalian cells contain riboflavins and other compounds which are naturally fluorescent and give the cell some level of autofluorescence. Many cells in tissue culture have higher levels of autofluorescence than those occurring in living tissue.
The level of autofluorescence of cells or particles can be measured by calibrating the particular fluorescence channel of interest in terms of MESFs and reading the value from the calibration plots as described in U.S. Pat. Nos. 4,714,682; 4,767,206; 4,774,189 and 4,857,451. The disclosure of all patents and patent applications cited herein including those referred to in the Cross-Reference to Related Applications, is hereby incorporated herein by reference.
Calibration of fluorescent microbead standards is provided by means of the invention of U.S. Pat. No. 4,714,682 which relates a microbead standard back to a stable and reproducible solution of primary standard which has the same excitation and emission spectra as the sample being measured. With sufficiently dilute solutions, considerations of quenching and changes of extinction coefficient may be avoided, as long as the spectra of the primary soluble standard solution, the microbead standards, and the labeled cells in the sample are the same. Thus, fluorescent intensities of a sample may be related to a quantitative concentration of a soluble primary standard via calibrated microbeads which have the same spectra.
As discussed in U.S. Pat. No. 4,714,682, fluorescenated microbeads may be calibrated with such a system in terms of Equivalent Soluble Dye Molecules per Microbead. For example, fluorescein microbeads are standardized against a primary laser grade fluorescein, which laser grade is stable, and of the highest purity of any of the fluorescein compounds and has excitation and emission spectra equivalent to that of FITC-labeled cells and the fluorescein microbead standards. The fluorescent microbead standards are calibrated by determining the fluorescent intensity of standard solutions of laser grade dyes with a fluorometer and relating those fluorescence intensities to the fluorescence intensity of suspensions of the microbeads. The number of microbeads in the suspension per unit volume is determined with a Coulter Counter.TM. or a Hemocytometer.TM.. Then from these data, the number of equivalent soluble dye molecules per microbead is calculated by dividing the equivalent soluble dye molecules per unit volume by the number of microbeads per that unit volume.
In co-pending application Ser. No. 07/374,435, the compensation circuits are adjusted such that the level of fluorescence in the fluorescence channels, other than the channel designated for particular fluorescent dye (the primary channel(s)), is equal to the level of fluorescence of the sample prior to labeling the sample with fluorescent dyes. Autofluorescent microbeads, matching the fluorescence spectra and intensity of the unlabeled, naturally fluorescent sample to be measured, are run on the flow cytometer in the FL1 versus FL2 fluorescence channel dot plot or histogram display mode.
The previous methods do not provide for qualitative method for determining machine sensitivity, and require use of quantitative standards and use of a calibration curve without providing information o the machine noise and sample autofluorescence.
It is therefore an object of the invention to provide a method of use of non-fluorescent particles to determine fluorescence threshold of a flow cytometer relative to the autofluorescence of samples.
Other objects and advantages of the invention will be more fully apparent from the following disclosure and appended claims.